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Abstract:

The invention provides a rabbit-derived immortal B-lymphocyte capable of
fusion with a rabbit splenocyte to produce a hybrid cell that produces an
antibody. The immortal B-lymphocyte does not detectably express
endogenous immunoglobulin heavy chain and may contain, in certain
embodiments, an altered immunoglobulin heavy chain-encoding gene. A
hybridoma resulting from fusion between the subject immortal B-lymphocyte
and a rabbit antibody-producing cell is provided, as is a method of using
that hybridoma to produce an antibody. The subject invention finds use in
a variety of different diagnostic, therapeutic and research applications.

Claims:

1-15. (canceled)

16. A hybrid cell made by fusing a rabbit spleen cell with a myeloma
cell, wherein: said rabbit spleen cell produces an antibody; said myeloma
cell does not detectably express rabbit IgH and immunoglobulin heavy
chain (IgH) mRNA expression of said myeloma cell is not detectable by
RT-PCR; and said hybrid cell produces said antibody.

17. The hybrid cell of claim 16, wherein said antibody specifically binds
to an target antigen.

18. The hybrid cell of claim 16, wherein said rabbit spleen cells are
harvested from a rabbit that has been immunized with an antigen.

19. The hybrid cell of claim 16, wherein said myeloma is a rabbit
myeloma.

21. The hybrid cell of claim 16, wherein said immunoglobulin heavy chain
has a sequence set forth as SEQ ID NO:2.

22. The hybrid cell of claim 16, wherein hybridoma cell is stable in
culture for more than one year.

Description:

FIELD OF THE INVENTION

[0001] The invention relates to a fusion partner for the production of
rabbit monoclonal antibodies.

BACKGROUND OF THE INVENTION

[0002] It has long been recognized that rabbit antibodies have favorable
properties compared to mouse antibodies. First, rabbits are known to
produce antibodies to many antigens that are poorly immunogenic in mice
(e.g., Norrby et al., Proc. Natl. Acad. Sci. 1987 84:6572-6; Raybould &
Takahashi, Science 1988 240:1788-90 and Weller et al., Development
1987;100:351-63). For example, Bystryn et al (Hybridoma 1982 1:465-72)
directly compared rabbit and mouse antibodies directed against human
melanoma cells and showed that they recognize different epitopes. Second,
rabbit antibodies are generally of high affinity. Third, because most
monoclonal antibodies are generated in mice and rats, relatively few
monoclonal antibodies are available that react with mouse or rat
immunogens. In spite of these advantages, until recently it has been
impossible to raise monoclonal antibodies in the rabbit. Monoclonal
antibodies are predominantly produced by using the hybridoma technology
originally developed by Koehler and Milstein (Nature 1975 256:495-7).
This method involves immortalization of antibody-producing lymphocytes by
fusion with myeloma cells. Since myeloma-like tumors are unknown in the
rabbit, it has been impossible to use this approach for producing rabbit
monoclonal antibodies. Attempts have been made to use mouse myeloma cell
lines as fusion partners (e.g., Raybould & Takahashi, 1988, supra), or to
use in vitro-transformed lymphoid cell lines from rabbits, but in all
cases these approaches were hampered by the instability of the resulting
hybridomas.

[0003] A novel approach was taken by K. Knight and colleagues
(Spieker-Polet et al., Proc. Natl. Acad. Sci. 1995 92:9348-52), who
succeeded in producing a myeloma-like tumor in transgenic rabbits
expressing oncogenes under the control of the immunoglobulin heavy and
light chain enhancers. They isolated a plasmacytoma cell line, termed
240E-1, from these tumors, and showed that fusions of 240E-1 cells with
rabbit lymphocytes produced hybridomas. Hybridomas were produced from
lymphocytes of rabbits immunized using plasma proteins or whole cells,
and were shown to secrete IgG antibodies.

[0004] An improved fusion partner cell line for the production of
monoclonal antibodies from rabbits is provided.

[0006] The invention provides a rabbit-derived immortal B-lymphocyte
capable of fusion with a rabbit splenocyte to produce a hybrid cell that
produces an antibody. The immortal B-lymphocyte does not detectably
express endogenous immunoglobulin heavy chain and may contain, in certain
embodiments, an altered immunoglobulin heavy chain-encoding gene. A
hybridoma resulting from fusion between the subject immortal B-lymphocyte
and a rabbit antibody-producing cell is provided, as is a method of using
that hybridoma to produce an antibody. The subject invention finds use in
a variety of different diagnostic, therapeutic and research applications.

[0008]FIG. 2 shows the amino acid sequence of the variable domain of the
endogenous heavy chain of 240E-W (SEQ ID NO:1), and its encoding cDNA
(SEQ ID NO:2). The endo-IgH cDNA was amplified by RT-PCR from 240E-W cell
mRNA, using primers corresponding to the 5' end of the rabbit vH segment,
combined with primers corresponding to the 3' end of the rabbit IgG
constant domain. The sequence of the mature vH domain is shown, beginning
with the first amino acid following the signal peptide.

[0009]FIG. 3 is a western blot showing that endo-IgH protein was not
detected in the 240E-W2 line, while it was strongly detected in the
240E-W. 240E-W2 cell lines do not express detectable endogenous Ig heavy
chain protein (endo-H chain). SDS-PAGE analysis of cell lysates prepared
from the parent fusion partner cell line, 240E-W (lane 1), and two
different 240E-W2 clones (lanes 2 and 3). Ig heavy chains were detected
by western blotting with goat-anti rabbit IgG antibodies. The 240E-W2
clones are derived from a hybridoma line that was subcloned and selected
for the absence of endo-H protein (see text). Note that Ig heavy chain
protein (50 kDa) is detected in the parent line 240E-W, but not in the
240E-W2 clones.

[0010]FIG. 4 contains two panels of gels showing genomic PCR
amplification products of 240E-W2 clones, and of 240E-W cells. The
results show that the gene encoding the endogenous IgH chain is
undetectable by genomic PCR in 240E-W2 cells. The 240E-W2 cell line does
not contain an intact gene encoding the endogenous Ig heavy chain
(endo-IgH). Genomic PCR was performed using primers specific for the gene
encoding the endogenous Ig light-chain (left-hand panel), or primers
specific for the endo-Ig heavy chain (left-hand panel). DNA was prepared
from different 240E-W2 subclones (lanes 1-18), or from the parent cell
line, 240E-W (lanes 19). Arrows denote the expected size of the genomic
fragment amplified by the primers that were used. Results show that the
endo-Ig light chain gene was equally detectable in both 240E-W and
240E-W2, whereas the endo-Ig heavy chain was only detected in 240E-W
(lane 19), and in DNA prepared from rabbit splenocytes (lane 6), but not
in any of the 240E-W2 clones. Faint bands of variable sizes are likely to
represent non-specific amplification products.

[0011] FIG. 5 shows a western blot demonstrating that hybridoma clones
derived from 240E-W2-C2 do not secrete any detectable endo-IgH. FIG. 5.
Rabbit hybridoma cell lines derived from the improved fusion partner cell
line, 240E-W2, secrete only one type of Ig heavy chain, and do not
produce the endogenous heavy chain present in 240E-W cells. Supernatants
from five different 240E-W2-derived hybridoma clones (lanes 1-3, 5, and
6), and a supernatant from a 240E-W-derived hybridoma (lane 4) were
analyzed by SDS-PAGE followed by western blotting with goat-anti rabbit
IgG antibodies. Note that the endo-IgH chain (lower band in lane 4) is
not detected in any of the 240E-W2-derived hybridoma supernatants,
confirming that the endo-IgH gene has indeed been removed or inactivated
in the 240E-W2 fusion partner cell line.

DEFINITIONS

[0012] The terms "antibody" and "immunoglobulin" are used interchangeably
herein. These terms are well understood by those in the field, and refer
to a protein consisting of one or more polypeptides that specifically
binds an antigen. One form of antibody constitutes the basic structural
unit of an antibody. This form is a tetramer and consists of two
identical pairs of antibody chains, each pair having one light and one
heavy chain. In each pair, the light and heavy chain variable regions are
together responsible for binding to an antigen, and the constant regions
are responsible for the antibody effector functions.

[0013] The recognized immunoglobulin polypeptides include the kappa and
lambda light chains and the alpha, gamma (IgG1, IgG2,
IgG3, IgG4), delta, epsilon and mu heavy chains or equivalents
in other species. Full-length immunoglobulin "light chains" (of about 25
kDa or about 214 amino acids) comprise a variable region of about 110
amino acids at the NH2-terminus and a kappa or lambda constant
region at the COOH-terminus. Full-length immunoglobulin "heavy chains"
(of about 50 kDa or about 446 amino acids), similarly comprise a variable
region (of about 116 amino acids) and one of the aforementioned heavy
chain constant regions, e.g., gamma (of about 330 amino acids).

[0014] The terms "antibodies" and "immunoglobulin" include antibodies or
immunoglobulins of any isotype, fragments of antibodies which retain
specific binding to antigen, including, but not limited to, Fab, Fv,
scFv, and Fd fragments, chimeric antibodies, humanized antibodies,
single-chain antibodies, and fusion proteins comprising an
antigen-binding portion of an antibody and a non-antibody protein. The
antibodies may be detectably labeled, e.g., with a radioisotope, an
enzyme which generates a detectable product, a fluorescent protein, and
the like. The antibodies may be further conjugated to other moieties,
such as members of specific binding pairs, e.g., biotin (member of
biotin-avidin specific binding pair), and the like. The antibodies may
also be bound to a solid support, including, but not limited to,
polystyrene plates or beads, and the like. Also encompassed by the terms
are Fab', Fv, F(ab')2, and or other antibody fragments that retain
specific binding to antigen.

[0016] An immunoglobulin light or heavy chain variable region consists of
a "framework" region interrupted by three hypervariable regions, also
called "complementarity determining regions" or CDRs. The extent of the
framework region and CDRs have been precisely defined (see, "Sequences of
Proteins of Immunological Interest," E. Kabat et al., U.S. Department of
Health and Human Services, (1983)). The sequences of the framework
regions of different light or heavy chains are relatively conserved
within a species. The framework region of an antibody, that is the
combined framework regions of the constituent light and heavy chains,
serves to position and align the CDRs. The CDRs are primarily responsible
for binding to an epitope of an antigen.

[0017] Chimeric antibodies are antibodies whose light and heavy chain
genes have been constructed, typically by genetic engineering, from
antibody variable and constant region genes belonging to different
species. For example, the variable segments of the genes from a rabbit
monoclonal antibody may be joined to human constant segments, such as
gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a
hybrid protein composed of the variable or antigen-binding domain from a
rabbit antibody and the constant or effector domain from a human antibody
(e.g., the anti-Tac chimeric antibody made by the cells of A.T.C.C.
deposit Accession No. CRL 9688), although other mammalian species may be
used.

[0018] As used herein, unless otherwise indicated or clear from the
context, antibody domains, regions and fragments are accorded standard
definitions as are well known in the art. See, e.g., Abbas, A. K., et
al., (1991) Cellular and Molecular Immunology, W. B. Saunders Company,
Philadelphia, Pa.

[0019] As used herein, the terms "determining," "measuring," and
"assessing," and "assaying" are used interchangeably and include both
quantitative and qualitative determinations.

[0020] The terms "polypeptide" and "protein", used interchangeably herein,
refer to a polymeric form of amino acids of any length, which can include
coded and non-coded amino acids, chemically or biochemically modified or
derivatized amino acids, and polypeptides having modified peptide
backbones. The term includes fusion proteins, including, but not limited
to, fusion proteins with a heterologous amino acid sequence, fusions with
heterologous and homologous leader sequences, with or without N-terminal
methionine residues; immunologically tagged proteins; fusion proteins
with detectable fusion partners, e.g., fusion proteins including as a
fusion partner a fluorescent protein, β-galactosidase, luciferase,
etc.; and the like.

[0021] As used herein the term "isolated," when used in the context of an
isolated antibody, refers to an antibody of interest that is at least 60%
free, at least 75% free, at least 90% free, at least 95% free, at least
98% free, and even at least 99% free from other components with which the
antibody is associated with prior to purification.

[0022] A "coding sequence" or a sequence that "encodes" a selected
polypeptide, is a nucleic acid molecule which is transcribed (in the case
of DNA) and translated (in the case of mRNA) into a polypeptide, for
example, in vivo when placed under the control of appropriate regulatory
sequences (or "control elements"). The boundaries of the coding sequence
are typically determined by a start codon at the 5' (amino) terminus and
a translation stop codon at the 3' (carboxy) terminus. A coding sequence
can include, but is not limited to, cDNA from viral, procaryotic or
eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and
synthetic DNA sequences. A transcription termination sequence may be
located 3' to the coding sequence. Other "control elements" may also be
associated with a coding sequence. A DNA sequence encoding a polypeptide
can be optimized for expression in a selected cell by using the codons
preferred by the selected cell to represent the DNA copy of the desired
polypeptide coding sequence.

[0023] "Encoded by" refers to a nucleic acid sequence which codes for a
polypeptide sequence, wherein the polypeptide sequence or a portion
thereof contains an amino acid sequence of at least 3 to 5 amino acids,
more preferably at least 8 to 10 amino acids, and even more preferably at
least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid
sequence. Also encompassed are polypeptide sequences that are
immunologically identifiable with a polypeptide encoded by the sequence.

[0024] "Operably linked" refers to an arrangement of elements wherein the
components so described are configured so as to perform their usual
function. Thus, a given signal peptide that is operably linked to a
polypeptide directs the secretion of the polypeptide from a cell. In the
case of a promoter, a promoter that is operably linked to a coding
sequence will direct the expression of a coding sequence. The promoter or
other control elements need not be contiguous with the coding sequence,
so long as they function to direct the expression thereof. For example,
intervening untranslated yet transcribed sequences can be present between
the promoter sequence and the coding sequence and the promoter sequence
can still be considered "operably linked" to the coding sequence.

[0025] By "nucleic acid construct" it is meant a nucleic acid sequence
that has been constructed to comprise one or more functional units not
found together in nature. Examples include circular, linear,
double-stranded, extrachromosomal DNA molecules (plasmids), cosmids
(plasmids containing COS sequences from lambda phage), viral genomes
comprising non-native nucleic acid sequences, and the like.

[0026] A "vector" is capable of transferring gene sequences to target
cells. Typically, "vector construct," "expression vector," and "gene
transfer vector," mean any nucleic acid construct capable of directing
the expression of a gene of interest and which can transfer gene
sequences to target cells, which can be accomplished by genomic
integration of all or a portion of the vector, or transient or
inheritable maintenance of the vector as an extrachromosomal element.
Thus, the term includes cloning, and expression vehicles, as well as
integrating vectors.

[0027] An "expression cassette" comprises any nucleic acid construct
capable of directing the expression of a gene/coding sequence of
interest, which is operably linked to a promoter of the expression
cassette. Such cassettes can be constructed into a "vector," "vector
construct," "expression vector," or "gene transfer vector," in order to
transfer the expression cassette into target cells. Thus, the term
includes cloning and expression vehicles, as well as viral vectors.

[0028] The term "specific binding" refers to the ability of an antibody to
preferentially bind to a particular analyte that is present in a
homogeneous mixture of different analytes. In certain embodiments, a
specific binding interaction will discriminate between desirable and
undesirable analytes in a sample, in some embodiments more than about 10
to 100-fold or more (e.g., more than about 1000- or 10,000-fold).

[0029] In certain embodiments, the affinity between a capture agent and
analyte when they are specifically bound in a capture agent/analyte
complex is characterized by a KD (dissociation constant) of less
than 10-6M, less than 10-7 M, less than 10-8 M, less than
10-9 M, less than 10-9 M, less than 10-11 M, or less than
about 10-12 M or less.

[0030] A polynucleotide is "derived from" a particular cell if the
polynucleotide was obtained from the cell. A polynucleotide may also be
"derived from" a particular cell if the polynucleotide was obtained from
the progeny of the cell, as long as the polynucleotide was present in the
original cell. As such, a single cell may be isolated and cultured, e.g.
in vitro, to form a cell culture. A nucleotide isolated from the cell
culture is "derived from" the single cell, as long as the nucleic acid
was present in the isolated single cell.

[0031] A "rabbit-derived" cell is a progenitor of a cell obtained from a
rabbit.

[0032] Before the present subject invention is described further, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited only
by the appended claims.

[0033] Where a range of values is provided, it is understood that each
intervening value, to the tenth of the unit of the lower limit unless the
context clearly dictates otherwise, between the upper and lower limits of
that range is also specifically disclosed. Each smaller range between any
stated value or intervening value in a stated range and any other stated
or intervening value in that stated range is encompassed within the
invention. The upper and lower limits of these smaller ranges may
independently be included or excluded in the range, and each range where
either, neither or both limits are included in the smaller ranges is also
encompassed within the invention, subject to any specifically excluded
limit in the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included limits are
also included in the invention.

[0034] Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Although any methods
and materials similar or equivalent to those described herein can be used
in the practice or testing of the present invention, the preferred
methods and materials are now described. All publications mentioned
herein are incorporated herein by reference to disclose and describe the
methods and/or materials in connection with which the publications are
cited.

[0035] It must be noted that as used herein and in the appended claims,
the singular forms "a", "and", and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example, reference to
"an antibody" includes a plurality of such antibodies and reference to "a
variable domain" includes reference to one or more variable domains and
equivalents thereof known to those skilled in the art, and so forth.

[0036] The publications discussed herein are provided solely for their
disclosure prior to the filing date of the present application. Nothing
herein is to be construed as an admission that the present invention is
not entitled to antedate such publication by virtue of prior invention.
Further, the dates of publication provided may be different from the
actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The invention provides a rabbit-derived immortal B-lymphocyte
capable of fusion with a rabbit splenocyte to produce a hybrid cell that
produces an antibody. The immortal B-lymphocyte does not detectably
express endogenous immunoglobulin heavy chain and may contain, in certain
embodiments, an altered immunoglobulin heavy chain-encoding gene. A
hybridoma resulting from fusion between the subject immortal B-lymphocyte
and a rabbit antibody-producing cell is provided, as is a method of using
that hybridoma to produce an antibody. The subject invention finds use in
a variety of different diagnostic, therapeutic and research applications.

[0038] In further describing the subject invention, the immortal
B-lymphocytes of the invention will be described first, followed by a
review of the methods and applications in which the subject cells find
use.

[0039] As mentioned above, the invention provides an immortal rabbit
B-lymphocyte fusion partner having undetectable endogenous immunoglobulin
heavy chain expression. The fusion partner cell may be fused with an
antibody-producing cell from a rabbit, e.g., an immunized rabbit, to
produce a hybridoma cell. The hybridoma cell, like the fusion partner,
produces no detectable endogenous immunoglobulin heavy chain, where
"endogenous" immunoglobulin heavy chain refers to the immunoglobulin
heavy chain encoded by the parental fusion partner cell.

[0040] In certain embodiments, therefore, the subject fusion partner may
contain an altered, non-functional, immunoglobulin heavy chain gene
(e.g., a heavy chain gene containing an insertion, deletion, inversion or
point mutation in the coding sequence or non-coding sequence such as in
the promoter or an intron of the heavy chain gene, for example; or the
heavy chain gene that may have been lost due to partial or complete
chromosome loss). In certain embodiments, the endogenous immunoglobulin
heavy chain polypeptide has the sequence shown in FIG. 2, or a variant
thereof that is at least 95%, e.g., at least 96%, at least 97%, at least
98%, or at least 99% identical to that sequence and may be encoded by the
cDNA shown in FIG. 2 or a cDNA that hybridizes to and/or is at least 95%,
e.g., at least 96%, at least 97%, at least 98%, or at least 99% identical
to that sequence. Immunoglobulin heavy chain gene expression may be
detected at the cDNA or protein level by RT-PCR or immunoblotting, for
example.

[0042] The fusion partner cell of the invention is further characterized
in that it is capable of fusion with a rabbit splenocyte at a highly
efficient rate (in the range of 40%-80%, or, in certain embodiments, at
least 80%), using methods essentially as described in Spieker-Polet et
al. (Proc. Natl. Acad. Sci. 1995 92:9348-52), but with the modifications
described in example 1. In other words, in a method that involves fusing
spleen cells of an immunized rabbit with the subject fusion partner at a
cell ratio of 2:1 (spleen cells:fusion partner cells), plating of those
cells in a 96-well microtiter plate at approximately 2×105
spleen cells per well and selecting of HAT-resistant cells, results in
approximately 40%-80%, or, in certain embodiments, at least 80% or at
least 90%) of the wells of the microtiter plate contain a hybridoma
colony.

[0043] Further to the above, the subject fusion partner is also
characterized by an ability to produce hybridomas that are stable (i.e.,
hybridomas that maintain the ability to produce a particular antibody for
an extended time periods, e.g., at least one year). In other words, a
monoclonal-antibody producing hybridoma cell line produced using a
subject fusion partner can be subcloned and subcultured for many
passages, until sufficient numbers of cells are obtained to produce IgG
antibody in amounts ranging from 1 milligram to 20 milligrams. In many
embodiments, the hybridoma cell line will be subcloned and subcultured to
reach a cell number of 30 million, and further subcultured in a
high-density culture system such as the Integra CellLine flask (BD
Biosciences). This typically allows the recovery of five to twenty
milligrams of IgG antibody from the hybridoma cell supernatant. In
particular embodiments, amount of antibody produced after one year of
culture will be at least 70%, at least 80%, at least 90%, or, in certain
embodiments at least 95% of the amount of antibody produced at the
beginning of the year.

[0044] Antibody-producing cells suitable for fusion with a subject fusion
partner include lymphocyte cells, e.g., a B lymphocyte. In a certain
embodiments, spleen cells from a hyperimmunized mammal, e.g., a rabbit,
are cultured with the subject fusion partner under conditions which allow
the cells to fuse. The hybridoma may be referred to a rabbit-rabbit
hybridoma. Such cell fusion methods are described in Spieker-Polet,
supra, and, as such, are well known in the art.

[0045] Further, and depending on exactly how the subject cell is made, the
cell may further contain a recombinant construct (e.g., a retroviral
vector inserted into the genome of the cell) adapted for expression of an
immortalizing (i.e., transforming) oncogene, i.e., a gene that allows a
cell to grow indefinitely in culture. Suitable oncogenes are well known
in the art (see, e.g., Katakura, Methods Cell Biol. 1998 57:69-91 for a
review). In one exemplary embodiment, the cell may express both the myc
and abl oncogenes, although a variety of other oncogenes may be readily
employed for the same purpose. Expression of the oncogene in the cell may
by directed by an immunoglobulin gene enhancer, e.g., the Eκ or
Eμ.

[0046] The subject fusion partner may be made by a variety of different
methods. In certain embodiments, the subject fusion partner may be made
by producing a parental fusion partner using a transgenic rabbit, and
then manipulating the parental fusion partner in culture to produce a
fusion partner having the desired characteristics. For example, a
suitable parent fusion partner may be made using known methods, e.g.,
those described in Spieker-Polet, supra, and that parental fusion partner
may modified by a number of means to produce the subject fusion partner.
In one embodiment, the parental fusion partner is a cell line called
240E-1, as described in U.S. Pat. No. 5,675,063 and Spieker-Polet, supra,
and deposited at the ATCC as accession no. HB-11870, or a cultured
thereof.

[0047] In one embodiment and as described in U.S. Pat. No. 5,675,063, a
transgenic rabbit is produced that contains a germ line insertion of an
oncogenic transgene, and expression of the transgene results in tumor
growth. A plasmacytoma (a tumor of B lymphocytes or plasma cells) forms
as a result of the tumor growth, and the plasmacytoma may be isolated
from the transgenic rabbit and cultured in vitro to produce a parental
fusion partner. The plasmacytoma cells may be irradiated in the presence
of 8-azaguanine to produce and select HAT sensitive mutants.

[0048] In another embodiment, a plasma cell may be isolated from a rabbit
and immortalized in culture via introduction of a construct for
expressing an oncogene. Such methods are standard in the art and reviewed
in Katakura (Methods Cell Biol. 1998 57:69-91).

[0049] Once a suitable parental fusion partner is obtained, the parental
fusion partner may be modified by a number of different means to produce
a subject fusion partner.

[0050] In one embodiment, the parent fusion partner may be repeatedly
sub-cloned to produce the subject fusion partner. Sub-cloning methods
usually involve culturing a population of cells to produce single
colonies, plating out cells from those single colonies onto separate
plates, and re-growing the plated cells into single colonies. Those
single colonies can be picked, tested for a desirable property and again
sub-cultured. Such methods are particularly suitable for use with
unstable cell lines or cell lines that have been mutagenized. For
example, in one embodiment, a parental fusion partner cell line can be
plated out to produce single colonies, and the single colonies tested for
production of endogenous immunoglobulin heavy chain, by RT-PCR or
immunological methods, for example. In certain embodiments, up to about
five and in certain embodiments up to about 10 rounds of sub-culturing
may be employed.

[0051] In another embodiment, a subject fusion partner may be made from a
parental fusion partner by making a targeted modification, e.g., a
targeted insertion, in the immunoglobulin heavy chain locus in the genome
of the fusion partner. Since the sequence of the rabbit germline
immunoglobulin locus is available (see, e.g., Ros et al, 2004 Gene 330,
49-59) and several fragments and rearranged versions thereof have been
deposited in NCBI's PubMed database, such targeted modifications may be
done using standard methods.

[0052] Once produced, the subject fusion partner may be used as a fusion
partner in any method known in the art. For example, a subject fusion
partner may be fused to a rabbit spleen cell to produce a stable
hybridoma. In one embodiment, a subject fusion partner may be fused to
spleen cells of an immunized rabbit to produce a plurality of hybridomas.
Those hybridomas may be screened to select a hybridoma producing a
monoclonal antibody of interest, and cultured to produce the antibody,
typically in the culture supernatant.

[0053] Procedures for immunizing animals are well known in the art as are
methods for the screening of hybrodimas and isolation of monoclonal
antibodies. For example, such methods are described in Harlow et al.,
(Antibodies: A Laboratory Manual, First Edition (1988) Cold Spring
Harbor, N.Y.) and other references cited throughout this disclosure.

[0054] The fusion partner cell line designated below as 240E-W2 was
deposited under the terms of the Budapest Treaty with the American Type
Culture Collection, 10801 University Blvd, Manassas, Va. 20110-2209, USA,
on BBBBB BBBBB, 2006 and assigned ATCC Accession No. BBBBBBB.

EXAMPLES

[0055] The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and description of
how to make and use the present invention, and are not intended to limit
the scope of what the inventors regard as their invention nor are they
intended to represent that the experiments below are all or the only
experiments performed. Efforts have been made to ensure accuracy with
respect to numbers used (e.g. amounts, temperature, etc.) but some
experimental errors and deviations should be accounted for. Unless
indicated otherwise, parts are parts by weight, molecular weight is
weight average molecular weight, temperature is in degrees Centigrade,
and pressure is at or near atmospheric.

[0056] The examples described below employ the materials and methods
described in Spieker-Polet (Proc. Natl. Acad. Sci. 1995 92:9348-52),
which reference is incorporated by reference herein in its entirety.

Example 1

[0057] In initial experiments, 240E-1 cells were fused with spleen cells
obtained from an immunized rabbit. Following HAT medium selection,
hybridoma colonies were isolated and tested for antibody production.
Several antibody-secreting hybridoma clones were obtained. After
expansion and repeated passaging of the hybridoma cell lines, it was
found that antibody secretion was greatly diminished in a majority of the
clones. Similar results were reported by others (Liguori et al., 2001,
supra). Furthermore, after subculturing the 240E-1 cells, their
efficiency as a fusion partner decreased. Similarly, Rief et al. (1998),
supra, reported a low yield of hybridomas, and only one out of six
positive hybridomas persisted after subcloning. The 240E-1 cell line is
therefore somewhat unstable, and impractical for routine use.

[0058] 240E-1 cells were subjected to repeated rounds of subcloning.
Subclones were selected for high cloning efficiency and robust growth, as
well as for morphological characteristics, such as a bright appearance in
phase contrast microscopy and formation of uniform adherent colonies.
Selected subclones were further tested for their ability to produce
stable hybridomas. After completing four rounds of this subcloning
process, a stable line (termed 240E-W) was obtained. The morphological
appearance of 240E-W cells differed significantly from the parent 240E-1
cells. 240E-W cells adhered more strongly to the substrate and did not
form clumps. Fusion of 240E-W cells with splenocytes of immunized rabbits
consistently yielded large numbers of stable hybridomas. In a typical
experiment, splenocytes from a rabbit immunized with mouse tumor cells
were fused with 240E-W cells at a ratio of 2:1, following the protocol
outlined in Spieker-Polet et al., 1995, with the following modification:
The cells were plated in 96-well plates, and HAT medium was added 3 days
after plating. After two weeks, 60% of the wells contained growing
colonies. Of these, 50% produced antibodies that reacted with the
immunogen. Upon expansion and subcloning, 80% of the clones grew
vigorously and retained antibody production over at least 5 passages.
Selected hybridoma clones were subcultured for up to one year without
decrease in antibody production. Hybridoma cell lines were exceptionally
robust and stable, and could be maintained in confluent wells for several
days without loss of viability.

Example 2

[0059] The 240E-W cell line and the original 240E-1 cell line, express a
significant amount of an endogenous IgG heavy chain (endo-IgH), which is
readily detectable at the protein and mRNA level (FIG. 3). The expression
of endogenous IgH persists in hybridomas derived from 240E-W, and is
often equal to or higher than the expression of the splenocyte-derived
IgH (spleno-IgH) comprising the heavy chain of the desired antibody
secreted by the hybridoma (FIG. 1). As shown in FIG. 1, endo-IgH and
spleno-IgH are, in most cases, readily distinguishable by SDS
polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence
and deduced amino acid sequence of endo-IgH was determined (FIG. 2), and
the identity of the lower band in FIG. 1 as endo-IgH was confirmed by
protein sequencing. Endo-IgH can associate with the splenocyte-derived Ig
light chain (spleno-IgL), as demonstrated by the presence of the intact
150 kDa IgG complex on non-reducing SDS-PAGE (data not shown). Therefore,
in many cases, hybridomas derived from 240E-W secrete a mixture of
endo-IgH/spleno-IgL and spleno-IgH/spleno-IgL. In a subset of hybridoma
clones, this may result in a loss of specificity and purity of the
hybridoma-derived antibody.

[0060] It was also found that a rearranged, potentially active, gene
encoding an endogenous kappa light chain (endo-IgL) is present in the
240E-W cell line, although the product of this gene could not be detected
on the mRNA or protein level. However, increased expression of the
endo-IgL gene was observed in a subset of 240E-W derived hybridomas. The
supernatants of these hybridoma cultures contained both endo-IgL and
spleno-IgL, in variable relative amounts (see FIG. 1). The nucleotide
sequence of endo-IgL was determined, and the identity of the endo-IgL
band shown in FIG. 1 was confirmed by protein sequencing. Thus, it was
shown that expression of endo-IgL is undetectable in the 240E-W fusion
partner cell line, but is detectable, at variable levels, in a subset of
hybridomas derived from 240E-W. This potentially affects antibody purity
and specific activity, due to the presence of mixed IgG tetramers
containing various combinations of endo-IgH, endo-IgL, spleno IgH, and
spleno-IgL.

Example 3

[0061] Fusion of 240E-W cells with rabbit splenocytes typically yields a
subset of hybridoma clones that do not secrete any detectable IgG. Since
even in the absence of secreted IgG the cells may contain intracellular
IgG heavy chains, several of these clones were tested by
immunocytochemistry for the presence of intracellular IgG. Briefly, this
was performed by culturing subclones in duplicate 96-well plates, and
processing one of the plates for immunocytochemistry by drying, fixation
with 0.4% paraformaldehyde, permeabilization with 0.5% Triton X-100, and
immunoperoxidase staining with polyclonal goat-anti-rabbit IgG
antibodies. Several clones were identified that contained only low levels
of IgG, and only in a subset of the cells. One of these hybridoma clones
was selected for subcloning and further testing. The cells were grown in
standard growth medium ((Spieker-Polet et al., Proc. Natl. Acad. Sci.
1995 92:9348-52), and subcloned by the limiting-dilution method.
Single-cell subclones were expanded and again tested for intracellular
IgG expression by immunocytochemistry. The subcloning was repeated twice,
until a clone was obtained that was 100% IgG-negative. This clone was
again subcloned, and it was found that all the subclones remained 100%
IgG-negative. In these subclones, IgH mRNA expression was undetectable by
RT-PCR. These clones were cultured in the presence of azaguanine (0.13
mM) for 3 weeks to select for revertants that had regained sensitivity to
HAT medium selection. Finally, two subclones were selected for further
analysis (240E-W2-C2 and 240E-W2-D1). These clones were analyzed by
genomic PCR, using primers specific for the endo-IgH sequence. As shown
in FIG. 4, the endo-IgH gene was not detected in the 240E-W2 subclones,
while it was strongly detected in the 240E-W cell line used as a positive
control. In contrast, the endo-IgL gene was equally detectable in 240E-W
and 240E-W2 (FIG. 4). This suggests that the 240E-W2 clones have
undergone a genetic deletion or rearrangement that resulted in the loss
of an intact endo-IgH gene.

[0062] The 240E-W2 subclones were further characterized with regard to
their utility as a fusion partner. Fusions were performed with
splenocytes collected from a rabbit immunized with a peptide derived from
human p53 protein, using the fusion partner cell lines 240E-W,
240E-W2-C2, or 240E-W2-D1. The 240E-W2-C2 cell line gave rise to
hybridomas that could be selected in HAT medium, and the fusion
efficiency (i.e., the percentage of wells containing hybridoma colonies)
was higher than the one obtained with 240E-W (82%-100% vs. 64%). In
contrast, the 240E-W2-D1 cell line gave rise to hybridomas at a low
efficiency (12%-22%). Further, the hybridoma wells derived from
240E-W2-C2 were shown by ELISA to contain antibodies reactive with the
immunogen, at a frequency similar to the one observed in the 240E-W
fusion. Several of these hybridomas were subcloned and used for in vitro
production of antibody, showing that hybridoma clones derived from
240E-W2 and 240E-W are similar in terms of stability and IgG
productivity. Taken together, these results demonstrate that the 240E-W2
cell line has the properties expected of an efficient fusion partner for
generating rabbit monoclonal antibodies.

[0063] SDS-PAGE analysis confirmed that hybridoma clones derived from
240E-W2-C2 do not secrete any detectable endo-IgH (FIG. 5). Expression of
endo-IgL was detected in only one of seven hybridoma clones derived from
240E-W2-C2. The removal of endo-IgH is likely to result in improved
antibody activity and specificity.

Example 4

[0064] Comparative studies were performed to evaluate 240E-W2 with 240E-W.
Both 240E-W and 240E-W2 were used as fusion partners to generate
hybridomas. Fusions were performed using conventional methodology.
1.5-3×108 lymphocytes from an immunized rabbit and the fusion
partner cells were mixed at a ratio of 2:1 with PEG 4000 at (Sigma P7181)
37° C. in serum-free medium. The cells were transferred into
96-well cell culture plates at approximately 1×105 lymphocytes
per well in 1640 RPMI medium with 15% FBS (or FCS), incubated in a
CO2 incubator. After 48 hr HAT medium (Sigma H0262) was added.
Hybridoma colonies were ready for screening in 3-5 weeks. Medium was
changed 2-3 times before screening by removing old medium and add fresh
medium. Supernatants were tested for the presence of antibody, specific
for the immunogen, by ELISA. Western blot analysis was used as a
secondary screening assay. The hybridomas were sub-cloned by limit
dilution. The fusion partner 240E-W at 2×104 cells per well
was used as feeder cells.

[0065] ELISA was performed in 96-well micro-titer plates that had been
coated overnight with immunogen at 1.0 μg/ml, plates were then
saturated with 2% BSA, followed by incubation with the
antibody-containing supernatant for 1 hr at room temperature.

[0066] After washing with PBS-Tween, alkaline phosphatase conjugated goat
anti-rabbit immunoglobulin (IgG) was added and incubation continued for
another hour; plates were washed again and developed in the presence of
P-NPP (para-nitrophenyl phosphate). Color was read at 405 nm with a plate
reader (Multiskan MCC/340 from Fisher Scientific).

[0067] Western blot analysis was performed by using whole cell lysates.
For example, breast cancer cell line (MCF-7) lysate (50 μg/lane) was
loaded on 10% SDS-polyacrylamide gel and proteins were separated by
electrophoresis. The proteins on the gel were transferred to
nitrocellulose membrane. The blots were blocked with 5% skim milk in
phosphate-buffered saline and incubated with the primary antibodies,
followed by HRP-conjugated goat anti-rabbit and mouse IgGs (Pierce,
20011). The blots were detected with ECL kit (Amersham RPN2106).

[0068] In 53 independent experiments, each for a different antibody, using
240E-W2 as the fusion partner cell, the average fusion efficiency
increased by 33%, the positive rates measured by ELISA and Western blot
analysis increased by 40% and 24% respectively; and the total success
rate for identifying suitable rabbit monoclonal antibodies increases by
41%, as compared with using 240E-W as the fusion partner cell.

[0069] A total of 35 hybridomas were subjected large scale production
using Integra expression system (DB 353137). In use of these hybridomas,
the average antibody yield increased by 32% and specific activities
measured by Western blot analysis increased by 133%, compared with
hybridomas generated using 240E-W as fusion partner cell.

[0070] It is evident from the above results and discussion that the
subject invention provides a important new fusion partner for the
production of rabbit monoclonal antibodies. Specifically, the subject
fusion partner allows highly efficient production of hybridoma cells that
are very stable and that produce no antibodies containing heavy chains
that are endogenous to the fusion partner. As such, the subject methods
and systems find use in a variety of different applications, including
research, therapeutic and other applications. The present invention
represents a significant contribution to the art.

[0071] While the present invention has been described with reference to
the specific embodiments thereof, it should be understood by those
skilled in the art that various changes may be made and equivalents may
be substituted without departing from the true spirit and scope of the
invention. In addition, many modifications may be made to adapt a
particular situation, material, composition of matter, process, process
step or steps, to the objective, spirit and scope of the present
invention. All such modifications are intended to be within the scope of
the claims appended hereto.